One third of all diabetic patients exhibit severe periodontal disease which is postulated to stem from an interplay of factors including increased susceptibility to infection, elevated pro-inflammatory cytokines, altered sensitivity to hormones, and hyperglycemia. The delayed regeneration of alveolar bone in the damaged periodontium reflects, in part, inadequate response to the anabolic actions of IGF-1 and insulin which may be down-regulated by cytokines and hyperglycemia. IGF-1 stimulation of osteoblasts, is anti-apoptotic and contributes to cell proliferation, differentiation, migration, matrix deposition and mineralization, and supports osteoclastogenesis. The specificity of the pleiotropic effects elicited by IGF-1R activation are mediated by the insulin receptor substrates, IRS-1 and IRS-2, which are critical to the formation of new bone and maintaining coupling between osteoblasts and osteoclasts. Signaling through the IRS proteins to PI3K/Akt and/or the Grb2/ERK pathways is mediated and modulated by posttranslational regulation. Perturbations in Tyr/Ser/Thr phosphorylation of IRS-1 occur in diabetes and osteoporosis. IRS-1 and IRS-2 are also glycosylated at Ser/Thr residues by N-acetylglucosamine (O-GlcNAc). This unique glycosylation is a reversible posttranslational modification proposed to regulate protein function in a manner analogous to phosphorylation. Using state-of-the-art tandem mass spectrometric approaches, we have identified multiple sites of O-GlcNAc modification on human IRS-1 and IRS-2 and developed methodology to measure the relative stoichiometry of this labile modification in a site-specific manner. The extent of protein O-GlcNAcylation changes rapidly in response to glucose, hormones, and cellular stress and it is implicated in altered insulin signaling and the glucose-induced complications associated with diabetes. In Aim 1, we propose to characterize the sites of O- GlcNAc modification of IRS-1 and IRS-2 in an osteoblast cell line and define the factors that modulate the extent of modification. We will investigate the interplay of phosphorylation and O-GlcNAc modification as these modifications may mutually exclude one another. The sites of O-GlcNAc modification of IRS-1 identified thus far are in close proximity to multiple SH2 domain binding motifs, thus in Aim 2 we will interrogate the effect of site-specific O-GlcNAc modification on interactions with the receptor and known binding partners and probe for novel protein interactions that may be induced by this modification. The role of the enzymes responsible for catalyzing the incorporation and removal of GlcNAc from proteins, O-GlcNAc transferase (OGT) and O- GlcNAcase (OGA), in modulating IGF-1 action in osteoblasts has not been addressed. Thus, in Aim 3, we will determine the impact of modulation of the expression of these enzymes on the cellular effects elicited by IGF-1 stimulation and whether altered O-GlcNAc modification of IRS-1 influences IGF-1 signaling in osteoblasts.